Hepatitis C virus (HCV) infects approximately 2% of the world's population and is the primary cause of liver transplants in the United States. Based on lessons learned from diseases such as AIDS, HCV RNA replication is a promising target for antiviral development. However, the replication of all viruses with plus-strand RNA genomes is poorly understood, especially at the biochemical level. The overall goal of the research in the Kao lab is to understand mechanism of RNA virus replication. [unreadable] The goal for this project is to build knowledge about the subunits of the HCV replication complex, with emphasis on protein-RNA, and protein-protein interactions. This is an extension of the past six years of research in the Kao lab, where a number of basic properties of the HCV polymerase and the HCV protease-helicase have been examined using biochemical, biophysical, and cell-based methods. The research can be partitioned into several related subaims that will: [unreadable] 1. Identify and validate the biological importance of the residues in the HCV RdRp that interacts with the substrate NTPs, the template RNA, and during different stages of HCV RNA synthesis. [unreadable] 2. Elucidate the interactions between the HCV RdRp and the nascent RNA and identify and characterize the nascent RNA exit channel. [unreadable] 3. Examine the protein-protein and protein-RNA interactions with other replicase- associated subunits of the HCV replicase and examine effects of the interactions on HCV replicase formation in cells. [unreadable] 4. Obtain images of the protein complexes using electron microscopy and reconstruct their structures. [unreadable] Results from this proposal will advance the understanding of the mechanism of viral RNA-dependent RNA synthesis for ALL positive-strand RNA viruses. The knowledge can also be used to compare the mechanisms of action of all template-dependent (both viral and cellular) polymerases. [unreadable] [unreadable] [unreadable]